1. Field of the Invention
The invention relates to the preparation and use of dendrimeric oligoguanidine on a tri-functional core effective for transporting bioactive substances into the cytosol of cells in vivo. The compositions contain guanidinium headgroups assembled around a tri-functional phosphoramide or phosphorodiamidate core.
2. Objects and Advantages
While considerable structural diversity is found among drugs and probe molecules which act on intracellular targets, the physical properties of most of these agents are restricted to a narrow range to ensure passage through the polar extra-cellular milieu and the non-polar lipid bilayer of the cell. This can be problematic in the field of drug discovery where the bioactive substances are impermeable or poorly permeable to the cells in animals. Cellular membranes are particularly impermeable to highly charged compounds such as polynucleotides, or neutral antisense macromolecules, such as Morpholino oligos and peptide nucleic acids. When a high molecular weight bioactive molecule (e.g. Morpholino oligo) is administered to an organism, its medicinal utility is generally limited by its inability to efficiently gain access to its intracellular target.
Researchers have attempted to develop technologies for enhancing the transport of chemical compounds across organismal barriers. For instance, Frankel et al. report the conjugation of selected molecules to HIV TAT protein (Frankel et al., PCT Pub. No. WO 91/09958 (1991)). Barsoum discusses the use of the HIV TAT peptide sequence RKKRRQRRR for enhancing transport across cellular membranes (Barsoum et al., PCT Pub. No. WO 94/04686 (1994)). Wender et al. discuss the use of oligoarginine moieties for increasing the delivery of various molecules across cellular membranes (Wender et al., US2003/0032593).
The enormous potential of arginine-based molecular transporters has stimulated efforts to develop improved structures for delivery of large polar molecules into cells. However, researchers have concluded from a large number of molecular transporters containing arginines in peptide or peptoid assembly, or guanidines in a variety of backbones, that (a) the guanidine headgroups are principally responsible for its uptake into cells, (b) backbone chirality is not critical for cellular uptake, and (c) the number of guanidine head groups between 7 and 15 is optimal for efficient uptake (Rothbard, J. B., et al. J. Am. Chem. Soc. 126:9506-9507 (2004)). Therefore, methods and compositions have been described for transporting drugs and macromolecules across biological membranes in which the drug or macromolecule is covalently attached to a transport polymer consisting of a scaffold containing oligoguanidines.
However, the practical application of such oligoguanidine transporters is generally limited due to their high cost and difficulty of use. Usually these oligoguanidines are prepared using a solid-phase synthesizer. Although this approach is readily automated and allows for the synthesis and purification of long oligomers, it suffers drawbacks including high cost, limited scalability, and the need for resin attachment and cleavage. In contrast, solution phase synthesis could avoid the cost and scale restrictions of resins. Despite numerous reports about the importance of guanidine groups in the peptidic backbone of oligoguanidines, most oligoguanidine delivery moieties have a linear structure, while relatively few attempts have been made using guanidine groups in branched architectures. In one such rare case, polyguanidino dendrimers using triamine-based diamino acid monomeric units were synthesized and their delivery efficacy out-performed an oligoarginine reference standard (Wender, P. A., et al. Organic Letters 7:4815-4818 (2005)). Branched-chain arginine peptides also have the ability to translocate through cell membranes and to bring exogenous proteins into cells (Futaki, S., et al. Biochemistry 41:7925-7930 (2002)). Dendrimeric oligoguanidines based on amino triol subunits are capable of translocation through the cell membrane (Chung, H.-H., et al. Biopolymers (Pept. Sci) 76:83-96 (2004)). An alternative architecture based on amino triacid scaffold demonstrated that the dendrimeric molecular transporters can not only enable transport of bioactive cargo across the cell membrane, but also control the delivery into defined intracellular compartments (Huang, K., et al. Bioconjugate Chem. 18: 403-409 (2007)).
However, the syntheses of the dendrimeric oligoguanidines mentioned above are lengthy or involve expensive reagents for their assembly. A need clearly exists for new compositions and methods offering superior performance in transporting compounds across biological barriers, as well as being more cost-effective to make and to link to their bioactive substances to be transported into cells. The present invention fulfills these and other needs.